Hog1 Activation Delays Mitotic Exit Via Phosphorylation of Net1

Hog1 activation delays mitotic exit via phosphorylation of Net1

Silvia Tognettia,b,1, Javier Jiméneza,1,2, Matteo Viganòa, Alba Ducha,b, Ethel Queraltc, Eulàlia de Nadala,b,3, and Francesc Posasa,b,3

aDepartament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, 08003 Barcelona, Spain; bInstitute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, 08028 Barcelona, Spain; and cCell Cycle Group, Cancer Epigenetics and Biology Program (PEBC), L’Hospitalet de Llobregat, Institut d’Investigacions Biomèdica de Bellvitge (IDIBELL), 08908 Barcelona, Spain

Edited by Douglas Koshland, University of California, Berkeley, CA, and approved March 11, 2020 (received for review October 19, 2019) Adaptation to environmental changes is crucial for cell fitness. In Finally, exit from mitosis appears to be promoted upon osmostress Saccharomyces cerevisiae, variations in external osmolarity trigger in late anaphase-arrested mutants via modulation of the release of the activation of the stress-activated protein kinase Hog1 the phosphatase Cdc14 (14). However, the effect of osmostress (high-osmolarity glycerol 1), which regulates gene expression, me- on early mitotic cells is still unclear. tabolism, and cell-cycle progression. The activation of this kinase In an unperturbed cell cycle, progression through mitosis de- leads to the regulation of G1, S, and G2 phases of the cell cycle to pends on Cdc14 (reviewed in refs. 18 and 19). The activity of prevent genome instability and promote cell survival. Here we Cdc14 is blocked from G1 to metaphase as a result of its binding show that Hog1 delays mitotic exit when cells are stressed during to the nucleolar protein Net1 (20, 21). Cdc14 release and acti- metaphase. Hog1 phosphorylates the nucleolar protein Net1, al- vation occur in two steps. The first drives Cdc14 relocalization tering its affinity for the phosphatase Cdc14, whose activity is from the nucleolus to the nucleus and depends on the activation essential for mitotic exit and completion of the cell cycle. The un- of Cdc5 (22–25) and FEAR (Cdc fourteen early anaphase re- timely release of Cdc14 from the nucleolus upon activation of lease) pathway (reviewed in refs. 18, 19, and 26). This initial Hog1 is linked to a defect in ribosomal DNA (rDNA) and telomere release is mediated by the activation of the Clb2–Cdc28 complex, segregation, and it ultimately delays cell division. A mutant of which phosphorylates Net1 on at least six sites, thereby desta- Net1 that cannot be phosphorylated by Hog1 displays reduced bilizing the Net1–Cdc14 complex (22, 27, 28). This release was viability upon osmostress. Thus, Hog1 contributes to maximizing recently reported to additionally depend on nucleolar ribosomal cell survival upon stress by regulating mitotic exit. DNA (rDNA) condensation (29). The functions of nuclear Cdc14 are essential for the establishment of a successful ana- cell cycle | mitosis | osmostress | Net1 | MAPK phase, and they include regulation of the anaphase spindle (30, 31), chromosome movements, and positioning of the anaphase – pon sudden environmental changes, cells must induce a nucleus (32) and segregation of rDNA (33 36) and telomeres Urapid and transient adaptive response to ensure survival. (37, 38). The second step, activated during late mitosis and The response to variations in extracellular osmolarity has been evolutionarily conserved, and it involves the activation of Significance mitogen-activated protein kinase (MAPK) signaling cascades. In Saccharomyces cerevisiae, the effector of the high-osmolarity Proper chromosome segregation is critical for the maintenance glycerol (HOG) pathway is the Hog1 MAPK, a functional ho- of genomic information in every cell division, which is required molog of p38 in higher eukaryotes. Upon phosphorylation, Hog1 for cell survival. Cells have orchestrated a myriad of control induces a cytoplasmatic response that acts on glycerol and ion mechanisms to guarantee proper chromosome segregation. transporters, metabolism, and translation. Additionally, Hog1 Upon stress, cells induce a number of adaptive responses to rapidly translocates into the nucleus, where it modulates tran- maximize survival that range from regulation of gene expres- scription to control gene expression and alters cell-cycle pro- sion to control of cell-cycle progression. We have found here gression (reviewed in refs. 1–3). that in response to osmostress, cells also regulate mitosis to The effect of osmostress on cell-cycle progression has been ensure proper telomeric and rDNA segregation during adap- addressed extensively in budding yeast (4–15). These studies tation. Osmostress induces a Hog1-dependent delay of cell- have unraveled a series of Hog1-dependent events that are finely cycle progression in early mitosis by phosphorylating Net1, tuned to prevent genetic instability and ensure maximal survival. thereby impairing timely nucleolar release and activation of Activation of the HOG pathway during each phase of the cell Cdc14, core elements of mitosis regulation. Thus, Hog1 acti- cycle leads to an alteration in the speed of progression, and the vation prevents segregation defects to maximize survival. mediators of this transient effect are phase-specific. Cells in G1 transiently arrest the cell cycle upon exposure to osmostress. This Author contributions: S.T., J.J., M.V., A.D., E.Q., E.d.N., and F.P. designed research; S.T., J.J., M.V., and A.D. performed research; S.T., J.J., M.V., E.Q., E.d.N., and F.P. analyzed data; and event is dependent on Hog1 in a dual manner: 1) stabilization of S.T., E.d.N., and F.P. wrote the paper. the cyclin-dependent kinase inhibitor Sic1 (10); and 2) down- The authors declare no competing interest. regulation of G1 cyclins via phosphorylation of the transcrip- This article is a PNAS Direct Submission. tion regulators Whi5 and Msa1 (10, 11, 16). The G1-to-S tran- This open access article is distributed under Creative Commons Attribution-NonCommercial- sition is also delayed via Hog1-induced transcriptional inhibition NoDerivatives License 4.0 (CC BY-NC-ND). of Clb5 (4), whereas S phase is regulated not only by delaying the 1S.T. and J.J. contributed equally to this work. accumulation of Clb5 and Clb6 (15) but also by directly acting on 2Present address: Department of Basic Sciences, Faculty of Medicine and Health Sciences, components of the replicative machinery such as Mrc1 (8). Hog1 Universitat Internacional de Catalunya, 08195 Barcelona, Spain. also impinges on the G2-to-M transition by down-regulating 3To whom correspondence may be addressed. Email: [email protected] or Clb2 expression and stabilizing Swe1, a negative regulator of [email protected]. Cdc28 whose degradation is required for entry into mitosis (5, 7, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ 17). Moreover, Hog1 controls the levels of a long noncoding doi:10.1073/pnas.1918308117/-/DCSupplemental. RNA on CDC28 to facilitate cell-cycle reentry upon stress (12). First published April 7, 2020.

8924–8933 | PNAS | April 21, 2020 | vol. 117 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1918308117 Downloaded by guest on September 24, 2021 dependent on Cdc14 nuclear localization, promotes the full re- morphogenesis) pathway, which leads to the transcriptional ac- lease of Cdc14 into the cytoplasm and relies on the MEN (mi- tivation of genes responsible for cell separation (46, 47), thereby totic exit network) (reviewed in refs. 39 and 40). As a result of ensuring timely septum disruption after cytokinesis (reviewed in MEN activation, Cdc14 is phosphorylated at sites adjacent to its refs. 48–50). nuclear localization signal and is consequently retained in the Here we show that the activation of Hog1 in metaphase leads cytoplasm (41). Cytoplasmatic Cdc14 directly promotes mitotic to delayed mitosis. This defect was not found to be linked to exit via dephosphorylation of the APC activator Cdh1, the mitotic spindle formation or elongation, or to nuclear division. In transcription factor Swi5, and the Cdc28 inhibitor Sic1. Addi- contrast, the timely release of Cdc14 was affected upon genetic tionally, cytoplasmatic Cdc14 is required for completion of mi- activation of Hog1. Hog1 phosphorylated the nucleolar protein tosis as it dephosphorylates a number of Cdc28 substrates, Net1 and thus negatively regulated Cdc14 release. Correspond- erasing the phosphorylation marks accumulated during the cell ingly, a Net1 unphosphorylatable mutant partially rescued the cycle (42–45). Among its cytoplasmatic targets, Cdc14 is also Cdc14 localization defect. Additionally, Hog1 activation resulted responsible for activating the RAM (regulation of Ace2 and in defective segregation of the late segregating regions (rDNA

WT 120 A B WT 120 100 sln1ts 100 sln1ts hog1 80 80 60 60

% cells 40 40 20 20 0 0 with anaphase% cells spindle 0 101520253035405060 0 1015202530 Time after release at 37°C (min) Time after release at 37°C (min) 0 15 30 140% sln1ts

120 CELL BIOLOGY 100 80 60

% cells C 40 100 *** * NS 90 20 80 0 70 * 0 101520253035405060 60 Time after release at 37°C (min) 50 WT 40 140% sln1ts hog1 30 sln1ts 120 20 sln1ts hog1 100 % cells with released Cdc14 10 0 80 05101520 60 Time after release at 37°(min)

% cells 40 010 20

0 0 101520253035405060 Time after release at 37°C (min) Control

Metaphase ts

Early Anaphase Metaphase Early Anaphase Late Anaphase/ Telophase hog1 sln1 ts

G1 sln1

Late Anaphase/ G1 Telophase

Fig. 1. Hog1 activation induces a defect in cell division and Cdc14 release in metaphase-arrested cells. (A) GAL1p-CDC20 cells were synchronized in metaphase in YPRaff at 25 °C for 3 h and switched to 37 °C for 1 h before release upon galactose addition. Nuclear dynamics were monitored by DAPI staining. Data represent mean and SD. Representative images of the WT strain show the temporal progression of nuclear division by DAPI staining. (B) Cells were treated as in A. Mitotic spindle length was measured by immunofluorescence (α-tubulin). Data represent mean of the percentage of cells with anaphase spindles (>2 μm) and SD. Representative microscopy images of the WT strain show how the mitotic spindles (α-tubulin, green) elongate over time in relation to nuclear division (DAPI, blue). (C) Cells were treated as in A. Cdc14 release was analyzed by fluorescence microscopy on fixed cells using strains bearing GFP- tagged Cdc14. Data represent mean and SD. Asterisks indicate the Student’s t test assuming unequal variance analysis comparing the WT with sln1ts (NS, no significance, P > 0.05; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005). Representative images correspond to Cdc14 signal at time 0 and 10 min after release at the restrictive temperature.

Tognetti et al. PNAS | April 21, 2020 | vol. 117 | no. 16 | 8925 Downloaded by guest on September 24, 2021 and telomeres), which was rescued by the Net1 unphosphorylatable Then, we attempted to determine which mitotic event(s) was mutant. Remarkably, this mutant is partially osmosensitive. Thus, impaired upon stress. Initially, we monitored the dynamics of Net1 is a target of Hog1 required to facilitate osmoadaptation nuclear division by DAPI staining of GAL1p-CDC20 cells re- during early stages of mitosis. leased from metaphase arrest at the restrictive temperature (Fig. 1A). In the WT strain, most of the cells at time 0 showed a Results clear metaphase phenotype, with one nuclear signal localized on Hog1 Activation Induces a Defect in Cell Division and Cdc14 Release in the mother cell. Over time, the percentage of binucleated ana- Metaphase-Arrested Cells. To study whether osmostress resulted in phase cells progressively increased, then augmenting the per- a delay after G2, we synchronized cells at early mitosis by means centage of late anaphase/telophase cells (separated nuclei with of expressing CDC20 under the control of the inducible galactose mother and daughter cells still attached to one another), to fi- promoter (GAL1p-CDC20), which arrested cells in metaphase. nally almost fully reach the typical G1 phenotype of separated The GAL1p-CDC20 expression system consists of the re- cells with a concentrated nuclear signal at 40 min (Fig. 1 A, Top). placement of the CDC20 promoter by the GAL1 promoter, and sln1ts nuclear distribution over time differed greatly from the thus cells arrest in metaphase in the absence of galactose and WT. Cells progressed normally through anaphase but, although reenter into the cell cycle again in the presence of galactose. This showing seemingly separated nuclei, failed to reach physical is a well-established and accepted tool for the synchronization of separation of mother and daughter (Fig. 1 A, Middle). Once ts cells when analyzing specific mitotic events (e.g., refs. 51 and 52). again, sln1 hog1 cells mimicked the behavior of the control The ability of cells to progress into a new cell cycle was analyzed strain (Fig. 1 A, Bottom). These data indicate that the activation by flow cytometry after release in control conditions or upon of Hog1 results in a delay in the late stages of mitosis. osmostress (SI Appendix, Fig. S1A). Control cells progressed A crucial step of successful mitosis is the establishment of a normally through mitosis and entered a new cell cycle 50 to functional spindle for chromosome segregation. To test whether 60 min after release. Remarkably, when released in the presence mitotic spindle stability or elongation was affected, cells carrying of 0.4 M NaCl, cells showed progression to a new G1 phase only GAL1p-CDC20 were synchronized and released at the restrictive temperature, and tubulin was immunostained to measure spindle after 90 min. Additionally, when mitosis progression was moni- ts ts tored by means of degradation of the mitotic cyclin Clb2, a delay length in WT, sln1 , and sln1 hog1 cells (Fig. 1B). All strains in Clb2 degradation was observed in osmostressed compared were observed to show a timely enrichment in the number of > μ with untreated cells (SI Appendix, Fig. S1B). These data indicate cells with anaphase spindles ( 2 m), thereby indicating that that osmostress induces a striking delay after release from early Hog1 activation does not impair mitotic spindle elongation. On the other hand, the persistence of longer spindles observed in mitosis. However, after an initial delay, cells were able to restore ts cell-cycle progression (SI Appendix, Fig. S1A), suggesting that sln1 cells suggests a delay in spindle disassembly. their viability is not compromised. GAL1p-CDC20 cells pro- The phosphatase Cdc14 is a master regulator of progression moted osmoadaptation upon stress similar to wild-type (W303) through mitosis (reviewed in refs. 56 and 57), and its localization cells (SI Appendix, Figs. S1C and S2C). Similar results were is modulated by interaction with the nucleolar protein Net1 (20, obtained when cells were synchronized using α-factor and re- 21). We thus assessed whether Cdc14 localization is impaired GAL1p-CDC20 leased in control conditions or stressed with 0.4 M NaCl when upon Hog1 activation. strains additionally bearing a enhanced GFP (yeGFP)-tagged version of endogenous entering mitosis (SI Appendix, Fig. S2 A and B). This is consistent Cdc14 were analyzed by fluorescence microscopy upon release with the idea that cells are able to adapt to the environmental from metaphase arrest at the restrictive temperature in WT, change and survive despite being exposed to stressful conditions. sln1ts, and sln1ts hog1 strains on fixed cells (Fig. 1C). The control Of note, cells expressing CDC20 from the MET3 promoter strain displayed a rapid release of Cdc14, which reached its [where Cdc20 expression was regulated by the presence of me- maximum 15 min after release from metaphase arrest. In con- thionine in the culture media (53)] also showed delayed pro- ts trast, the sln1 strain failed to initiate Cdc14 release as promptly. gression into a new cell cycle upon stress when compared with Additionally, successful relocalization of Cdc14 in this strain control conditions (SI Appendix, Fig. S2D), thereby indicating occurred in only ∼71% of the cells compared with ∼92% of that the phenotype observed upon osmostress does not depend control cells at the same time point (15 min after release). sln1ts on the synchronization method. hog1 cells mimicked the pattern of the control strain. These To verify the involvement of the MAPK pathway in the ob- observations indicate that Hog1 activation impairs the timely served phenomena upon osmostress after release from early release of Cdc14. Given the crucial function of this phosphatase mitosis synchronized cells, we compared a GAL1p-CDC20 strain in mitotic exit, this finding could explain the delay observed in (from here on, WT) with a strain additionally bearing a cell-cycle progression. temperature-sensitive SLN1 allele (sln1ts), which leads to con- stitutive genetic activation of Hog1 independent of external Hog1 Phosphorylates Net1 In Vitro and In Vivo. Upon stress-induced stimuli when exposed to the restrictive temperature (54, 55). It is activation, the MAPK cascade, through its effector Hog1, is re- worth mentioning that activation of the HOG pathway by genetic sponsible for a rapid alteration in the phosphoproteome of the manipulation has served to unravel the direct role of Hog1 in the cell to mediate an effective response to environmental changes cell cycle (4, 7, 8, 10, 15). Cell-cycle progression and Clb2 protein (58). We found that Cdc14 release is impaired upon genetic dynamics were monitored under these conditions. Consistent activation of Hog1 and thus tested whether this phosphatase is a with what was observed upon salt addition, genetic activation of direct target of Hog1. Cdc14 phosphorylation was assessed in an Hog1 promoted a delay in the progression into a new G1 phase in vitro kinase assay using glutathione S-transferase (GST)-tag- for cells released from metaphase arrest at 37 °C (SI Appendix, ged versions of Cdc14, Hog1, and its activator Pbs2 (in its con- Fig. S1D). This delay was also reflected in a defect in full timely stitutively active form, Pbs2EE) purified from Escherichia coli degradation of Clb2 in the sln1ts strain (SI Appendix, Fig. S1E). (Fig. 2A). Under the experimental conditions tested, Hog1 did Remarkably, for both cell-cycle progression and Clb2 degrada- not phosphorylate Cdc14. However, Cdc14 release has been tion and reaccumulation, HOG1 deletion in the sln1ts back- reported to depend on its binding to the nucleolar protein Net1 ground fully suppressed the delay caused by Hog1 activation (SI and the initial release is in fact promoted by Clb2–Cdc28– Appendix, Fig. S1 D and E). Taken together, these data indicate dependent phosphorylation on Net1 (22, 28). We then hypothesized that Hog1 mediates a delay when cells are released from that Hog1 acts directly on Net1 to negatively regulate the release of early mitosis. Cdc14. To test for Net1 phosphorylation, we performed an in vitro

8926 | www.pnas.org/cgi/doi/10.1073/pnas.1918308117 Tognetti et al. Downloaded by guest on September 24, 2021 A GST-Hog1/Pbs2EE -+ +- N terminus of the protein (a fragment we called F2). Strikingly, GST-Cdc14 + + + + this region contains the Cdc14-binding domain (amino acids 1 to 341) (59) and Clb2–Cdc28 phosphorylation sites responsible for EE Pbs2 - promoting Cdc14 release (22, 28). The Net1 F2 sequence contains Cdc14 - Cdc14 several S/T-P sites that can be phosphorylated by Hog1. All these * serine and threonine residues were mutated to alanine, alone or in Hog1 - combination, expressed as recombinant GST–Net1 F2, and tested Coomassie Autorad. for Hog1 phosphorylation in vitro as done previously (SI Appen- dix,Fig.S4). The combination of alanine mutations on T62 and S385 almost fully abolished the phosphorylation signal in com- B parison with the WT (Fig. 2C). GST-Hog1/Pbs2EE -+ +- To validate Net1 as a target of the osmostress-induced response, GST-Net1 ++ + + we tested its phosphorylation status in vivo. GAL1p-CDC20 strains bearing tandem affinity purification (TAP)-tagged endogenous Net1 - Net1 Net1 were synchronized in metaphase, cultures were divided into two groups, and one was stressed with 0.4 M NaCl for 5 min (SI * Pbs2EE - Appendix,Fig.S3C). Net1 mobility shift was analyzed on Phos-tag gels in WT cells and hog1 and Net1T62A,S385A mutants (Fig. 2D). In Coomassie Autorad. response to osmotic shock, the control strain showed a reduced mobility shift of Net1 signal when compared with the untreated sample. Importantly, no clear differences between the treated and C untreated hog1 and Net1T62A,S385A mutant strains were observed. 1goH-TSG - ++ - ++ This result suggests that, upon osmotic stress, Net1 is phosphory- GST-Pbs2EE + ++ + ++ latedinvivobyHog1onresiduesT62andS385. GST-Net1 F2 T62A,S385A --+ --+ GST-Net1 F2 ++ - ++ - Hog1-Dependent Phosphorylation of Net1 Impairs Cdc14 Release. Since Hog1 phosphorylates Net1 and activation of this kinase alters Cdc14 release, we envisioned that these phosphorylations Pbs2EE - Net1 F2 - Net1 F2 could affect the stability of the Net1–Cdc14 complex. To address CELL BIOLOGY Hog1 - this question, we tested whether Hog1-dependent phosphorylation of Net1 interferes with the capacity of Clb2–Cdc28 to Coomassie Autorad. modify Net1 itself, thus affecting Cdc14 release. To this end, we performed a two-step in vitro kinase assay in which GST–Net1 D F2 was incubated sequentially with activated Hog1 and Clb2–Cdc28 (SI Appendix, Fig. S5A). The Clb2–Cdc28 complex phosphorylated Net1 in vitro, regardless of Hog1, indicating that Hog1 does not prevent Clb2–Cdc28 phosphorylation on Net1. T62A, S385A To rule out that the residues required for phosphorylation of WT hog1 Net1 Hog1 and Clb2–Cdc28 could be the same, Net1 F2 wild type and -+-+-+0.4 M NaCl T62A, S385A were tested in an in vitro kinase assay with Clb2–Cdc28 (SI Appendix, Fig. S5B). Clb2–Cdc28 could phos- Net1-TAP phorylate the alanine mutant, suggesting that Clb2–Cdc28 and Hog1 phosphorylations on Net1 occur on different residues. Next, the interaction between Net1 and Cdc14 was recon- Phostag gel stituted in vitro in a multistep binding assay in order to re- capitulate the contribution of Clb2–Cdc28 and Hog1 to the Fig. 2. Hog1 phosphorylates Net1 on T62 and S385 in vitro and in vivo. (A) stability of the complex (Fig. 3 C and D and schematic repre- Bacterially expressed GST-tagged Cdc14 was used as substrate for the in vitro kinase assay with GST–Hog1. Degradation/contaminating bands are in- sentation in Fig. 3 A and B). Briefly, purified Cdc14 and Net1 F2 dicated with an asterisk. (B) Bacterially expressed GST–Net1 was tested as proteins from E. coli were allowed to interact, and the complex substrate of GST–Hog1 in vitro. Degradation/contaminating bands are in- was retained on an affinity resin. To promote the disassembly of dicated with an asterisk. (C) Comparison of the phosphorylation signal of the the complex, samples were then incubated with Clb2–Cdc28 wild-type fragment F2 of Net1 (amino acids 1 to 511) with the fragment immunoprecipitated from metaphase-arrested yeast cells. To test mutated to alanine on T62 and S385 in an in vitro kinase assay with whether Hog1 induced stabilization of the Net1–Cdc14 complex, GST–Hog1. (D) GAL1p-CDC20 cells bearing TAP-tagged endogenous Net1 purified Hog1 and Pbs2EE from E. coli were added to the re- were synchronized in metaphase in YPD and harvested in the control con- action prior to Clb2–Cdc28. Samples were extensively washed to dition or after a 5-min treatment with 0.4 M NaCl. Protein extracts were release all unspecifically bound proteins and analyzed by West- analyzed in a 6% polyacrylamide gel containing 10 μM Phos-tag, and Net1 mobility was followed by Western blot (α-TAP). ern blot (Fig. 3C). Finally, the relative % of Net1 binding was quantified (Fig. 3D). The unspecific binding of Net1 to the resin was tested in the absence of Cdc14 (Fig. 3C, sample 1). The assay kinase assay with GST-tagged Net1 (Fig. 2B). Net1 was phosphory- performed with unmodified Net1 F2 showed the interaction between Net1 and Cdc14 (Fig. 3 A, C, and D, sample 2). Addi- lated by Hog1, thereby indicating that Net1 is a direct substrate tionally, as expected, the addition of Clb2–Cdc28 caused a of the kinase. To further explore Net1 as a substrate of Hog1, we marked destabilization of the complex (decreased GST–Net1 screened the functional domains of Net1 (SI Appendix, Fig. S3A) binding; Fig. 3 A, C, and D, sample 3), an observation consistent and generated a series of GST-tagged truncations of the protein, with the findings of a previous study (22). However, of note, which were tested for in vitro phosphorylation (SI Appendix, Fig. when Hog1 was added to the reaction, the interaction between S3B). These analyses showed that the Hog1-dependent phos- Net1 and Cdc14 appeared to be stabilized, even in the presence phorylation sites were present in the first 511 amino acids of the of Clb2–Cdc28 (Fig. 3 A, C, and D, sample 4). These results thus

Tognetti et al. PNAS | April 21, 2020 | vol. 117 | no. 16 | 8927 Downloaded by guest on September 24, 2021 A C 6His-Cdc14 - ++-++ + + GST-Net1 F2 + ++--- + - GST-Net1 F2 T62A, S385A --- - +++ + Clb2-9Myc - -+--+ + + GST-Hog1/Pbs2EE -----+ + - Pbs2EE - Net1 F2 - α-GST Hog1 -

Cdc14 - α-HIS

Clb2 - α-Myc

123568 4 7

D * NS

* 200

NS 150 *** ** 100

B 50 Relative % Net1 binding 0 6His-Cdc14 - ++-++ + + GST-Net1 F2 + ++--- + - GST-Net1 F2 T62A, S385A --- - +++ + Clb2-9Myc - -+--+ + + GST-Hog1/Pbs2EE -----+ + -

E 100 NS NS * 80

60 ****

sln1ts 40

sln1ts Net1 T62A, S385A 20 % cells with released Cdc14 with released % cells sln1ts hog1

0 0 5 10 15 20 Time after release at 37°C (min)

Fig. 3. Hog1-dependent phosphorylation of Net1 impairs Cdc14 release. (A and B) Schematic representation of the in vitro reconstitution experiment for Net1–Cdc14 binding. Briefly, purified Cdc14 and Net1 F2 proteins (unmodified or Net1T62A,S385A) were allowed to interact, and the complex was retained on an affinity resin. To promote the disassembly of the complex, samples were then incubated with Clb2–Cdc28. Hog1/Pbs2EE were added to test whether Hog1 induced stabilization of the Net1–Cdc14 complex. (C) In vitro reconstituted Net1–Cdc14 interaction using purified proteins was monitored by Western blot, probing for Net1 (α-GST), Cdc14 (α-HIS), and Clb2 (α-Myc). (D) Quantification of the in vitro binding assays. Data represent mean and SD; asterisks indicate statistically significant differences by the Student’s t test assuming unequal variance (NS, no significance, P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.005). (E) Cells were treated as in Fig. 1A. Cdc14 release was analyzed by fluorescence microscopy on fixed cells using strains bearing GFP-tagged Cdc14. Data represent mean and SD. Statistical analysis comparing sln1ts with sln1ts Net1T62A,S385A. Asterisks indicate statistically significant differences by the Student’s t test assuming unequal variance (NS, no significance, P > 0.05; *P ≤ 0.05, ****P ≤ 0.001).

indicate that Hog1 is indeed involved in modulating Cdc14 re- F2T62A,S385A did not promote stabilization of the Net1–Cdc14 lease. To further confirm the critical role of Hog1 phosphory- complex (Fig. 3 B–D, sample 8), thereby demonstrating that lation on Net1 residues T62 and S385, the assay was then Hog1 does phosphorylate the two residues of Net1 to impair performed using the Net1 F2T62A,S385A mutant (Fig. 3 C and D Cdc14 release. Of note, the presence of Hog1/Pbs2 alone did not and schematic representation in Fig. 3B). The reconstitution of alter Net1–Cdc14 complex stability (neither for the wild type nor the complex with Cdc14 was similar to that achieved with the for the alanine mutant) (SI Appendix, Fig. S5 C and D). To study wild-type Net1 (Fig. 3 B–D, sample 6 compared with sample 2), whether Hog1-dependent phosphorylation of the residues T62 indicating that the two sites are not required to promote or and S385 of Net1 is sufficient to cause the observed impairment sustain binding. Furthermore, the introduction of Clb2–Cdc28 of Cdc14 release (Fig. 1C), we monitored Cdc14 localization in resulted in the destabilization of the Net1–Cdc14 complex to a sln1ts, sln1ts Net1T62,S385A, and sln1ts hog1 strains upon release similar extent as that of wild-type Net1 (Fig. 3 B–D, sample 7 from metaphase arrest at the restrictive temperature, as pre- compared with sample 3). These data show that the modification viously described (Fig. 3E). The sln1ts strain showed a clear delay of residues T62 and S385 per se does not interfere with the role in Cdc14 release when compared with sln1ts hog1. The sln1ts of Clb2–Cdc28 in facilitating Cdc14 release from Net1. Impor- Net1T62A,S385A strain showed overall higher levels of nucleolar tantly, prior addition of Hog1 to the reaction involving Net1 release compared with sln1ts and promoted a prompt increase in

8928 | www.pnas.org/cgi/doi/10.1073/pnas.1918308117 Tognetti et al. Downloaded by guest on September 24, 2021 Cdc14 release already at 5 min after exit from metaphase arrest, at 30 min after release, and full release was not reached in ∼30% as occurred in the sln1ts hog1 strain. of the cells. These observations thus indicate that Hog1 activation Altogether, these data indicate that Hog1-dependent phos- has an impact on rDNA segregation. phorylation of Net1 is required for the stabilization of Cdc14 Telomere segregation was monitored on a similar set of strains binding and thus the prevention of Cdc14 release. bearing TetR-YFP tetO:1061Kb-ChrXII (Fig. 4 C and D). In this case, the dynamics of segregation were slower and less homo- Hog1 Activation during Mitosis Impairs rDNA and Telomere geneous among cells of the same population, with the control Segregation. The early functions of Cdc14 include the pro- strain reaching ∼80% segregation only at 60 min. Of note, sln1ts motion of rDNA and telomere segregation, so-called late seg- Net1T62A,S385A and sln1ts hog1 reached a similar extent of segre- regating regions, as they divide after all other genomic regions gation. In contrast, the sln1ts strain was unable to fully segregate have segregated (33–35, 37, 38). To test whether the segregation the telomeric region of chromosome XII during the time course. of these regions is affected upon activation of Hog1, we per- The segregation of the telomeric region upon genetic activation formed fluorescence time-lapse microscopy on GAL1p-CDC20 of Hog1 was also assessed in cells reaching mitosis from α-factor strains bearing a TetO/TetR system, where a yellow fluorescent block/release (SI Appendix, Fig. S6 A and B). Consistently, the protein (YFP)-tagged reporter could be recruited to a specific sln1ts strain displayed a clear defect in telomere segregation genomic locus (60, 61). rDNA segregation was monitored over compared with sln1ts hog1 and this defect was almost fully re- time after release from metaphase arrest at the restrictive tem- versed in the sln1ts Net1T62A,S385A mutant strain. This observation perature on WT, sln1ts, sln1ts Net1T62A,S385A,andsln1ts hog1 strains therefore indicates that Hog1 phosphorylation of Net1 affects bearing TetR-YFP tetO:450Kb-ChrXII (Fig. 4 A and B). The control the segregation of this genomic region. Additionally, the muta- strain started segregating the rDNA region at around 20 min after tions T62A and S385A on Net1 did not have any effect on release, reaching ∼80% segregation at 30 min. As expected, the telomere segregation in a SLN1 wild-type background (SI Ap- sln1ts Net1T62A,S385A and sln1ts hog1 strains showed similar behav- pendix, Fig. S6C). Taken together, these data indicate that ge- ior. Strikingly, the sln1ts strain started to segregate the rDNA only netic Hog1 activation impairs the segregation of rDNA and

A WT sln1ts Net1 T62A, S385A Time after release at 37°C (min) B sln1ts sln1ts hog1

0 (DIC) 0 10 20 30 40 CELL BIOLOGY 100 WT 80

sln1ts 60

40 sln1ts % cells with Net1 T62A, S385A 20 unsegregated rDNA

ts 0 sln1 hog1 0 102030405060708090 Time after release at 37°C (min)

C Time after release at 37°C (min) D WT sln1ts Net1 T62A, S385A 0 (DIC) 0 10 20 30 40 sln1ts sln1ts hog1

WT 100

80 sln1ts 60

ts sln1 40 Net1 T62A, S385A % cells with 20

ts sln1 hog1 unsegregated telomere 0 0 102030405060708090 Time after release at 37°C (min)

Fig. 4. Hog1 activation during mitosis impairs rDNA and telomere segregation. (A) GAL1p-CDC20 TetR-YFP tetO:450Kb-ChrXII cells were synchronized in metaphase in YPRaff at 25 °C for 3 h and switched to 37 °C for 1 h before release upon galactose addition. rDNA segregation was monitored by time-lapse microscopy. DIC, differential interference contrast. (B) Quantification of rDNA segregation (as in A). Data represent mean and SD. (C) GAL1p-CDC20 TetR-YFP tetO:1061Kb-ChrXII cells were synchronized in metaphase in YPRaff at 25 °C for 3 h and switched to 37 °C for 1 h before release upon galactose addition. Telomere segregation was monitored by time-lapse microscopy. (D) Quantification of telomere segregation (as in C). Data represent mean and SD.

Tognetti et al. PNAS | April 21, 2020 | vol. 117 | no. 16 | 8929 Downloaded by guest on September 24, 2021 telomeres. To measure whether these segregation defects com- promise genomic integrity, we measured Rad52-YFP foci to assess the presence of recombination foci. Net1T62A,S385A cells displayed significantly higher levels of Rad52 foci compared with A 2.5 Control (YPGal) Net1 wild-type cells (SI Appendix, Fig. S6D). Finally, to assess whether segregation defects could ultimately affect bud-neck 2 structures, we monitored by time-lapse microscopy the localiza- 1.5 tion of Myo1, a protein involved in bud-neck morphogenesis and WT cytokinesis (SI Appendix, Fig. S6 E and F). The sln1ts strain re- OD660 1 leased at the restrictive temperature displayed a clear defect in Net1T62A, S385A Myo1 localization compared with the WT. This delay in the 0.5 hog1 actomyosin ring disassembly could be accountable for the pronounced defect, in contrast with the delay observed in 0 6 48 66 54 60 24 12 18 30 Cdc14release,incell-cyclecompletionobserveduponHog1 36 42 activation. Time (h)

Hog1-Dependent Phosphorylation of Net1 Is Required for Osmoadaptation. 2.5 2.5 1 M NaCl (YPGal) Hog1 activation in metaphase-arrested cells induces a delay in cell-cycle progression that appears to be linked to Net1 phos- 22 phorylation on residues T62 and S385. We thus tested whether the phosphorylation of Net1 is required to maximize cell survival 1.51.5 upon osmostress. To this end, WT, Net1T62A,S385A, and hog1

strains were presynchronized in metaphase and released either in OD660 11 yeast extract peptone galactose (YPGal) or in YPGal supple- 0.5 mented with 1 M NaCl. Cell growth was monitored for 66 h after 0.5 release in the two conditions (Fig. 5A). All strains were equally 00

able to grow in YPGal (control), thereby indicating that neither 6 60 12 18 24 54 66 HOG1 deletion nor T62 and S385 mutations on Net1 affected 30 36 42 48 cell fitness. When grown in media containing NaCl, the WT Time (h) strain adapted over time and reached normal growth. As expected, the hog1 strain did not proliferate under these conditions as it lacked the capacity to adapt to high osmolarity. B Control (YPGal) 0.8 M NaCl (YPGal) T62A,S385A Strikingly, the Net1 strain showed osmosensitivity WT compared with the WT strain, thereby indicating that the T62 and S385 residues of Net1 are involved in osmoadaptation dur- Net1T62A, S385A ing mitosis. To corroborate this finding, we also tested the same hog1 set of strains for growth on solid YPGal media in the presence or absence of 0.8 M NaCl upon release from metaphase arrest (Fig. 5B). Once again, the WT strain grew in NaCl, whereas the condition was lethal for the hog1 strain. Similar to what was observed in liquid media, the Net1T62A, S385A strain showed less C Anaphase growth than the WT cells on the NaCl plate. The reduced viability of the unphosphorylatable mutant of Net1 was also ob- Clb2 Cdc14 rDNA/telomeres Cdc28 served in asynchronous cultures (SI Appendix, Figs. S7 and S8 A P PP segregation and B), confirming its disadvantage for growth upon osmostress. Net1 Of note, Net1T62A,S385A cells also displayed defective growth upon osmostress compared with a W303 wild-type background Cdc14 CELL (SI Appendix, Fig. S8C). Altogether, these data show that Hog1 DIVISION Net1 phosphorylation of Net1 on T62 and S385 is required for proper adaptation to high osmolarity. Cdc14 IMPAIRED PP P P P rDNA/telomeres Discussion Clb2 Cdc28 Net1 segregation A key feature of living beings is their capacity to adapt to environmental changes and thus ensure maximal cell survival. Mi- tosis is a particularly vulnerable moment of the cell cycle Hog1 involving the onset of a series of critical events, such as sister DELAYED CELL DIVISION chromatid segregation, cytokinesis, and physical separation of Osmostress mother and daughter cells. Defects in chromosome segregation and cell division can have dramatic effects on cell fate. For in- Fig. 5. Hog1-dependent phosphorylation of Net1 is required for osmoa- stance, in human cells, DNA missegregation can lead to genetic daptation. (A) GAL1p-CDC20 cells were synchronized in metaphase in YPRaff instability and tumorigenesis (62, 63). Therefore, upon pertur- and released via galactose addition in YPGal (control) or in the presence of bation of the extracellular environment, cell-cycle regulation 1 M NaCl (osmostress). Growth was monitored at 25 °C every 30 min in a must take place during all phases of the cycle, including mitosis, 96-well plate using a Synergy H1 MultiMode Reader. Data represent mean and SD. (B) GAL1p-CDC20 cells were synchronized in metaphase in YPRaff to ensure cell fitness. and released via galactose addition before spotting fivefold dilutions on Upon cellular stress, the G1, S, and G2 phases of the cell cycle – YPGal or YPGal-containing 0.8 M NaCl plates. (C) Schematic representation are delayed (4, 5, 7, 8, 10 12, 15). In this study, we found that of Hog1-mediated osmoadaptation during mitosis. Hog1 phosphorylates metaphase cells exposed to osmostress also showed a Hog1- Net1 residues T62 and S385, impairing Cdc14 release. As a result, rDNA and dependent delay in cell-cycle progression. Additionally, the telomere segregation are affected, causing a delay in cell division.

8930 | www.pnas.org/cgi/doi/10.1073/pnas.1918308117 Tognetti et al. Downloaded by guest on September 24, 2021 dynamics of Cdc14 release from the nucleolus were delayed nuclei but might instead arrest with residual genomic material upon genetic activation of the kinase Hog1. Cdc14 is a master belonging to the late segregating regions stretched in-between regulator of mitosis and its localization-dependent activation is mostly separated nuclei. Additionally, studies that analyzed the crucial for cell-cycle completion (reviewed in refs. 39 and 64). phenotype of thermosensitive mutants of Cdc14 (cdc14-1 and Cdc14 nucleolar release during anaphase is promoted by cdc14-3) at the restrictive temperature reported cell-cycle arrest Clb2–Cdc28–dependent phosphorylation on the N-terminal re- in late anaphase/telophase, with generally separated nuclear gion of Net1 (22, 27). We therefore hypothesized that Hog1 masses but unsegregated telomeres and rDNAs (34, 35, 60). The alters the Net1–Cdc14 complex association via phosphorylation phenotype observed in the event of Hog1 genetic activation of one of the two proteins. We found that, at least in vitro, Hog1 during metaphase, with initial impairment of the timely release phosphorylated Net1 on residues T62 and S385, which are in of nucleolar Cdc14 and cells arrested in late mitosis, therefore close proximity to the Cdc14-binding domain (59). Consistently, resembles that reported in the literature for thermosensitive in vitro reconstitution of the Net1–Cdc14 interaction indicated Cdc14 mutants. In contrast, the delay in metaphase observed in that Hog1 interferes with the ability of Clb2–Cdc28 to promote the present study differs from what was observed when mitotic disruption of the complex. In an unperturbed cell cycle, timely cells bearing MEN mutants were driven to exit mitosis in re- regulated accumulation of Clb2–Cdc28 facilitates Cdc14 release, sponse to hyperosmotic stress (14). We propose that this ap- which in turn promotes dephosphorylation of a number of tar- parent discrepancy lies in the differential biochemical state in gets and segregation of late genomic regions to finally allow for which cells encounter stress. MEN mutant cells should arrest in cell division. We speculate that, upon activation during mitosis, anaphase, presumably after Cdc14 has already been transiently Hog1 phosphorylates Net1 on residues T62 and S385 and, by this released from the nucleolus into the nucleus as a function of means, transiently increases Net1 affinity for Cdc14 rendering FEAR activation (reviewed in refs. 18, 19, and 26). This condi- the Net1–Cdc14 complex more resistant to dissolution upon tion is strikingly different from the metaphase arrest/release Clb2–Cdc28 phosphorylation. This event affects Cdc14 timely analyzed in our study, in which cells still have Cdc14 bound to release and in turn rDNA/telomere segregation, ultimately Net1 and sequestered into the nucleolus. Under the conditions delaying cell division (see the schematic diagram in Fig. 5C). We analyzed in their study (14), Hog1 might overrule the MEN re- therefore propose that cells execute a program to ensure quirement for Cdc14 release in order to maximize survival. Ac- osmoadaptation during early stages of mitosis via Hog1- cordingly, Reiser and colleagues (14) found that the hypertonic dependent phosphorylation of Net1 on residues T62 and S385. environment per se does not accelerate exit from mitosis and An important question regards the biological reason un- that cells treated with sorbitol progressed through mitosis less derlying a transient arrest of cell-cycle progression during the efficiently than untreated cells. These data are fully consistent CELL BIOLOGY early stages of mitosis upon osmostress. Previously reported with our observations that mitotic exit is delayed in cells that face osmoadaptive responses that led to a delay in the transition an osmotic shock before or during metaphase (with Cdc14 still through other cell-cycle phases could be explained by the need sequestered in the nucleolus) and that Net1 is involved in the for the cell to prevent or regulate DNA replication (G1 and S process of osmoadaptation. However, we find plausible that phase, respectively) and postpone mitosis (G2) to avoid sub- Hog1 may phosphorylate targets other than Net1 to modulate optimal execution of these crucial steps while adapting to envi- cell-cycle progression in mitosis. ronmental change. When cells are forced to adapt to osmostress At present, we also cannot rule out that the direct action of while already committed to division, as occurs in metaphase, they Cdc14 on promoting segregation of the late segregating regions might sustain the osmoadaptive program to maximize survival. is paired with additional functions of the phosphatase on other Therefore, a delay in mitosis might be required in order to factors required for successful mitotic exit (i.e., Aurora B, as prevent defects in DNA segregation. In fact, the Net1 mutant reported in ref. 35). On the other hand, upon hyperosmotic that failed to delay Cdc14 release became osmosensitive. Al- shock, Hog1-dependent phosphorylation of Net1 could not only though Cdc14 requirement and functions appear to differ slightly impair Cdc14 release but also alter other functions of Cdc14 or between organisms, all living beings most likely require the de- Net1, functions that could be related to the stability of the nu- phosphorylation of Cdk substrates to end a cell cycle and enter a cleolus and rDNA silencing, or to additional activities of the new G1 phase. Of note, human Cdc14B rescues cdc14 lethality in phosphatase Cdc14. In the absence of additional evidence, we budding and fission yeast, thereby suggesting functional conser- speculate that the Cdc14-related defect in full timely sister vation (65, 66). Interestingly, disruption of the Cdc14B locus in chromatid segregation could be reflected in a delay in organized human cells causes an increased incidence of rDNA anaphase septum formation by physical means, having unsegregated bridges, thus pointing to a defect in segregation, despite no ap- stretches of DNA lying in-between mother and daughter cells parent impairment of cell-cycle progression (67). Given the in- and ultimately obstructing proper septum formation. The exact creased complexity of human compared with budding yeast cells, mechanisms by which Cdc14 release, together with rDNA and a scenario in which additional factors complement or facilitate telomere segregation defects, mediates the delay in cell-cycle Cdc14 functions can be envisioned. progression deserves further attention. Budding yeast Cdc14 promotes the dephosphorylation of In summary, here we report that activated Hog1 phosphory- many proteins that are phosphorylated by cyclin-dependent ki- lates Net1, thus impairing Cdc14 nucleolar release and rDNA nases throughout the cell cycle (reviewed in refs. 42–44 and 56). and telomere segregation, ultimately delaying cell-cycle pro- It has been proposed that, in addition to Cdc14 activation, the gression. Here we identify a regulatory mechanism, through Clb2–Cdc14 balance is important for execution of mitotic exit phosphorylation of Net1, by which cells maximize survival via the events (68, 69). Along this line, even nondramatic changes in the regulation of mitosis upon environmental changes. timing or in the number of Cdc14 molecules released can have noticeable effects on cell-cycle progression. Among the most Materials and Methods extensively studied effects of Cdc14 release is the promotion of rDNA and telomere segregation via inhibition of transcription Synchronization of GAL1p-CDC20 cells in all experiments was achieved by – incubating exponential cells in yeast extract peptone raffinose (YPRaff) for 2 for completion of the cell cycle (33 35, 38). Consistently, here we to 4 h and released by inducing Cdc20 expression by galactose addition (1%). report that Hog1 activation during metaphase not only causes an Activation of the HOG pathway was achieved by incubating cells at the in- impairment in Cdc14 nucleolar release but is also linked to a dicated NaCl concentration or by genetic means using the sln1ts allele at defect in the timely segregation of the late segregating regions. 37 °C. For immunofluorescence (IF) and DAPI staining experiments, cells

This finding suggests that these cells do not have fully divided were fixed overnight with 3.7% formaldehyde in IF buffer (0.1 M K2HPO4,

Tognetti et al. PNAS | April 21, 2020 | vol. 117 | no. 16 | 8931 Downloaded by guest on September 24, 2021 0.1 M KH2PO4, 0.5 mM MgCl2, pH 6.4). Cells were then washed twice with IF kinase buffer (SI Appendix) and 100 μM ATP. After 30 min at 30 °C, 2 μgof T62A,S385A buffer and once with sorbitol buffer (0.1 M K2HPO4, 0.1 M KH2PO4,0.5mM GST–Cdc14, GST–Net1, GST–Net1 F2, and GST–Net1 F2 was added to EE 32 MgCl2, 1.2 M sorbitol, pH 7.4). Each sample was digested in sorbitol buffer the Hog1/Pbs2 mixture together with [ P]ATP (0.1 mCi/mL; PerkinElmer) with 2 μL β-mercaptoethanol and 40 μg/μL zymolase T100 (AttendBio) for and incubated for 30 min at 30 °C. Full lists of reagents, strains, and plasmids 30 min at 30 °C. For Cdc14 localization analysis, samples were collected every and detailed protocols used in this article are provided in SI Appendix, 5 min after release at 37 °C and fixed for 10 min on ice with 3.7% formal- Methods. dehyde in phosphate-buffered saline (PBS). Cells were spun down, resus- pended with a solution of 1% formaldehyde in PBS, and incubated for Data Availability. All data discussed in this study are included in the main text 10 min before extensive washes with 40 mM Tris (pH 8.8). For rDNA and and SI Appendix. telomere segregation analysis in metaphase-arrested cultures, cells were synchronized at the onset of metaphase, switched to 37 °C, and let attach to eight-well chambers pretreated with Con A, and unattached cells were ACKNOWLEDGMENTS. We thank Dr. Josep Clotet and Dr. Jordi Torres-Rosell for strains; and Dr. Berta Canal and Dr. Gerhard Seisenbacher for helpful washed away before release by galactose addition. For α-factor synchroni- discussions and suggestions. S.T. was the recipient of a contract for zation, cultures were arrested with 20 μg/mL α-factor for 3 to 4 h at 25 °C Investigador Doctor Junior (PDJ 2014, Generalitat de Catalunya) and a Juan and released at 25 °C for 60 min before switching to 37 °C to promote ge- de la Cierva fellowship. The study was supported by grants from the Spanish netic activation of Hog1 in mitosis. During this time, cells were let attach as Ministry of Economy and Competitiveness (PGC2018-094136-B-I00 to F.P.; before. For Myo1 localization analysis, cells were synchronized at the onset BFU2017-85152-P and FEDER to E.d.N.; BFU2016-77975-R and FEDER to E.Q.), of metaphase, switched to 37 °C, and let attach to eight-well chambers the Catalan Government (2017 SGR 799), and Fundación Botín, by Banco pretreated with Con A, and unattached cells were washed away before Santander through its Santander Universities Global Division (to F.P.). We analysis. In either case, images were acquired every 10 min for at least gratefully acknowledge institutional funding from the Spanish Ministry of 90 min at 37 °C with a 100× oil objective lens in the YFP or Cherry spectrum. Economy, Industry and Competitiveness (MINECO) through the Centres of Analysis of electromobility shifts in Phos-tag gels was achieved in a 6% Excellence Severo Ochoa Award, and from the CERCA Programme of the polyacrylamide gel supplemented with 10 μM Phos-tag (Wako) and 20 μM Catalan Government and the Unidad de Excelencia María de Maeztu, MnCl2 for 3 h at 100 V on ice. Basic Hog1 kinase assays were performed using funded by the AEI (CEX2018-000792-M). F.P. is the recipient of an ICREA 1 μgofGST–Hog1 activated with 0.5 μgofGST–Pbs2EE in the presence of Acadèmia Award (Generalitat de Catalunya).

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